Tuberculosis protein structure defined

Researchers in the Department of Biophysics and Biophysical Chemistry at the Hopkins School of Medicine have discovered the structure of a protein integral to the drug resistance of Mycobacterium tuberculosis infections. This information can provide essential insight into drug design that would inhibit the function of this protein and hopefully increase treatment successes.

Tuberculosis is a common yet deathly infectious disease that typically attacks the lungs and is transmitted by a bacterium called Mycobacterium tuberculosis (Mtb). In 1993, the World Health Organization declared TB a “global health emergency.” As the infection enters the lungs, it travels to the alveoli, which is the last stop for air to exchange oxygen and carbon dioxide with the blood. There, it replicates within the cells that reside in the alveoli, gradually invading the lung and potentially travelling to other parts of the body. Symptoms associated with TB include fevers, chills, weight loss, fatigue and finger clubbing.

Multidrug resistant strains of Mtb have been a major challenge for doctors to treat, as they negate the effects of drugs administered to infected patients. In fact, one of the greater challenges of treating tuberculosis is to eradicate the one percent of bacteria that survived the first week of treatment. They are known as persisters, able to change themselves to a deactivated state and slow their cell machinery down significantly.

“The greater problem arises when symptoms recede, and patients tend to stop complying with their drug needs,” Mario Bianchet, assistant professor of neurology and one of the co-discoverers, said.

These particularly robust bacteria can form unique bonds in their cell walls that effectively shelter their cell body. The researchers were able to deduce the structure of the protein responsible for catalyzing these bonds using a technique known as x-ray crystallography. This is a widely used technique that determines the electron density, or arrangement of atoms, of compounds by shooting a beam of x-rays and analyzing the diffraction patterns. Essentially, by back-tracing the position and intensity of the diffraction patterns, researchers are able to identify very specific locations where x-rays diffracted off of certain atoms. Using this method, the researchers were able to determine the structure of a protein known as Ldt Mt2, which is able to create special bonds in the cell wall.

Typically, bacteria have a layer around their wall called peptidoglycan, which is a mesh-like blanket of interlocking sugars and amino acids. The most common type of bond that interlinks each component is called D,D 4-3, a name that identifies the position of the bond relative to the structure of the amino acids. Penicillin, the once-called miracle drug, binds to proteins that catalyzes these D,D 4-3 linkages that in effect destroys the protective covering of bacteria.

Another type of linkage is the L,D 3-3 linkage, which is catalyzed by the protein of interest, Ldt Mt2. These linkages are highly elusive of drug effects that typically block enzyme functions that produce 4-3 linkages. 3-3 linkages were first identified in 1974 by Juana Wietzerbin; however, it was only until the protein Ldt Mt2 was implicated in the drug resistance of tuberculosis that the biological significance of these linkages was recognized.

Afterwards, it was a major underlying goal of Mario Amzel’s lab in the Department of Biophysics and Biophysical Chemistry to determine the structure of Ldt Mt2 and to propose a catalytic mechanism that induces these unique linkages. With their newly published 1.7 angstrom resolution of the protein structure, they provided a crucial stepping stone to designing high-affinity drugs that can inhibit the function of Ldt Mt2, thereby eliminating the drug resistance of tuberculosis. “[It] will enable us and others to design inhibitors of this L-D transpeptidase that may shorten the treatment of TB by being effective against the persistent mycobacterium” states Amzel.

Beyond fighting TB infections, the structure of this protein may pave way for strategies to fight against other pathological agents, including Enterococcus faecium and Clostridium difficile.

“In the future, we hope to study a family of related enzymes that are expressed in different times of the cell’s life cycle,” Bianchet said. This may lead to greater insight to periods in the life cycle when certain drugs are most effective.